Team:Tianjin/Integrated HP
—————————— Part A ——————————
Why "CREATE"
How did we decide to make chromosome-free yeast and come up with which needs or values should be prioritized in our project's design？
Ⅰ: Starting with the basics
A scientific project can never develop in isolation; it has to be continuously shaped by a variety of individuals in order to have a real impact on society. In view of this, we have fully considered the needs of target groups and our responsibilities to them in the process of project development. We wanted to ensure we not only finished with a sound scientific product, but with something that could actually make the world a better place. In the following paragraphs you will be able to follow our progress, thought processes, and how we have integrated experts' concerns to make our project a better one.
Ⅱ: Why did we decide to make CREATE?
Through reviewing papers, we have learned about the current cutting-edge technologies of synthetic biology and some unsolved cutting-edge problems. The cell-free system has aroused our great interest. Since then, we have discussed for many times and extensively consulted papers on prokaryote chromosome free system, minicell, vesicle and other related aspects, which greatly inspired us. Finally, we decided to create an unprecedented "cell"-chromosome free yeast. For cells, it is subversive to have no chromosomes, so we decided to have a bold try, and our idea was supported by literature research, and our teacher also affirmed it. The selection of eukaryotic cells for operation is in consideration of the superiority of eukaryotic cells in protein expression, which will lay a good foundation for our subsequent application and promotion.
—————————— Part B ——————————
Increase formation rate
After confirming the use of chromosome-free eukaryotic cells as our project, we began to design and conduct pre-experiments. In the course of the experiment, some things bothered us: 1. What method do we choose to cut yeast chromosomes. 2. How to increase the formation rate of chromosome-free cells. In order to solve these problems, we plan to interview relevant experts in the next step to listen to their suggestions.
Ⅲ: What method should we choose to cut chromosomes?
At the very beginning of our project, we need to find effective method for degrading the yeast's chromosome. At the beginning, we chose restriction endonucleases to cut chromosomes, but we encountered the problems of low cutting efficiency and low formation rate of chromosome-free cells. In order to solve this problem, we interviewed skilled users of CRISPR Dr. Yali Cui. According to her suggestions, we conducted extensive discussions about which cutting method to choose and finally decided to use CRISPR to cut yeast chromosomes. Through this method, our cutting efficiency has been greatly improved.
Ⅳ: How to improve the formation rate?
When we have gotten CREATEs, we found that we were not very efficient at getting chromosomal yeast cells, which means that we need to find methods to improve the efficiency of yeast transform and increase the formation rate of chromosomal free cell. After communicating with Associate Professor Yingxiu Cao, who mainly engaged in the development of synthetic biology tools, we listened to her suggestions and improved the new plasmid by adding a CRE transposon to the original GAL promoter and used β-estrogen to induce its expression.
Ⅴ: Hold CRISPR Conference
In the process of project promotion, We have some problems in the construction process, especially with CRISPR. When we found way to solve them, we realize that other teams may also encounter problems when using CRISPR. In order to solve our problems, provide an idea for other teams to solve problems, and provide reference for future iGEM teams, we organized CRISPR conference. Eleven iGEM teams actively participated in our interactive session where we shared our CRISPR problems and shared our ideas and drafted CRISPR Handbook, which are beneficial to both igemers and global interest.If you want to get more content, please click read more.
—————————— Part C ——————————
Practical applications
In fact, we think that the very existence of chromosome-free yeasts is a huge breakthrough. And every breakthrough in scientific research will bring practical applications. We need to know what our project can bring to the world. We come up with the idea of using the CREATEs to produce poisonous products, deliver drugs and deliver large DNA fragments. We'll test our ideas through experiments, which will be groundbreaking.
Ⅵ: Survey on college students
The ideas of students are endless, unlike experts, their ideas are not limited by professional knowledge, their ideas are unconstrained and creative. Therefor, after constructing CREATEs, we hoped to get feedback of college students on our project by issuing a questionnaire, and to understand their expected applications of CREATE. In the process of questionnaire collection, we have widely understood their expectations for our project. Through the collection of the questionnaire and analysis of the results, and our comprehensive consideration, we have determined the following applications: Producing poisonous drugs, drugs delivery and transform large DNA fragments.
So the next step, we intended to obtain more information through expert interviews, explore whether the public's expectations can be turned into reality.
Ⅶ: Integrated concerns from experts
Unconstrained ideas require expertise to realize. In order to fully consider the interaction between the world and our project, and timely adjust the project progress and make reasonable decisions. We investigated the views of experts and enterprises in relevant fields on the application of chromosome-free yeast. Also, the researchers from CPU put forward some suggestions for us. Their feedback not only shed light on potential areas with high demand but also pivoted us away from applications that our technology might not be suited for. For each possible application, we interviewed experts in each field to support us. Through interviews, we try our best to ensure the safety of project application and the feasibility of experimental operation.
Drug producing
Aim
In order to test whether this application is feasible, we had an online meeting with the researchers in Bota Bio, which can let us know the advantages and disadvantages of our project in practical production. Meanwhile, we visited Prof. Hao Song, who is the director of pharmaceutical engineering in Tianjin Universifty.
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Drugs delivery
Aim
In order to understand the existing methods of drug delivery and the possibility of using CREATE for drug delivery, we interviewed Prof. Yumiao Zhang, an expert in drug delivery. He introduced us a lot of information about drug delivery and pointed out the advantages and disadvantages of CREATE in drug delivery.
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Transform large DNA fragments
Aim
We asked doctors in lab who were engaged in DNA scientific research about their views of our project in order to gain some information about the limits of large DNA fragments transformation and what we need to do to improve our project.
After analyzing the information they gave us and various measurements, we finally learned that although our CREATEs has great potential in this application, it may not be the best choice at present.
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Others
We enquired what other expectations about our project in contributing to the world by discussing with professors and scientific researchers. We know other possibilities about our project and take them into account after communication:
△Bio-senser: Detect the concentration and pH value of a certain ion in the body. Or by monitoring the microenvironment in vivo, cancer cells may be detected early.
△It can be used to characterize the role of a single or several genes.
Through interview and communication, we identified the potential application prospect of high demand and these information help us avoid making detours. We finally chose to produce drugs as the future application direction of our project. In order to estimate whether the application we got after trade-offs are feasible to our project or not, we conceptualized experiments utilizing. If you want to get more content, please click read more.
—————————— Part D ——————————
Safety and ethicality
In order to develop a truly impactful technology, it was important for us to consider the safety and ethicality of our work.
Ⅷ: Social Research by questionnaire
We have prepared questionnaires for different audiences, hoping to understand the ideas of different people on the safety of CREATEs and biological in vivo therapy.
By this way, we understand the views of potential users on our project, so as to obtain the direction of project improvement. Meanwhile, the questions raised by the public also prompted us to find more experts in relevant fields to solve problems. Here are our questionnaires and analysis.
Ⅸ: Feasibility and safety: scientific experts' opinions
In response to the public's doubts about our project, we also conducted self-reflection. We have always thought that without chromosomes, microorganisms are safe, but is that really the case? To this end, we interviewed the experts of the Center of Biosafety Research and strategy-Dr. Xinyu Song to understand our responsibility to comply with ethics and how we fulfill it.
According to Dr. Xinyu Song’s suggestion, more biosafety issues will be considered in the follow-up of our project. For the follow-up development of the project, we plan to prepare from two aspects: for scientific researchers and for people who are not engaged in biological work. Ask the scientific researchers about the significance of the project, get research guidance and suggestions; popularize the science of bioengineering to the public, and get the general recognition of the public. We hope that with the popularization of bioengineering, more and more people will understand synthetic biology, the management of synthetic biology will become more and more standardized, biosafety will be paid more and more attention, and people's acceptance of biological products will also increase higher and higher.
Ⅹ: COP15 youth biodiversity science exploration activities
Because of the potential of biosafety demonstrated by our project, we were invited to participate in COP15 youth biodiversity science exploration activities sponsored by Publicity and Education Center of the Ministry of Ecological Environment. We shared our project to participants and undertook a small part of the drafting of Declaration on responsible scientific and technological innovation of Chinese synthetic biology youth to help biodiversity conservation (Draft) . If you want to get more content, please click read more.